1. Field of the Invention
The present invention relates to a novel T1Receptor (T1R)-like ligand II protein. In particular, isolated nucleic acid molecules are provided encoding the T1R-like ligand II protein. T1R-like ligand II polypeptides are also provided, as are recombinant vectors and host cells for expressing the same. This invention further relates to pharmaceutical compositions and formulations comprising T1R-like ligand II. Also provided are methods of using T1R-like ligand II polynucleotides, polypeptides, antibodies or agonists/antagonists for therapeutic and diagnostic purposes. Diagnostic kits are further provided.
2. Related Art
Interleukin-1 (IL-1).
Interleukin-1 (IL-1xcex1 and IL-1xcex2) is a xe2x80x9cmultifunctionalxe2x80x9d cytokine that affects nearly every cell type, and often in concert with other cytokines or small mediator molecules. (Dinarello, C. A., Blood 87:2095-2147 (Mar. 15, 1996).) There are three members of the IL-1 gene family: IL-1xcex1, IL-1xcex2, and IL-1 receptor antagonist (IL-1Ra). IL-1xcex1 and IL-1xcex2 are agonists and IL-1Ra is a specific receptor antagonist. IL-1xcex1 and xcex2 are synthesized as precursors without leader sequences. The molecular weight of each precursor is 31 kD. Processing of IL-1xcex1 or IL-1xcex2 to xe2x80x9cmaturexe2x80x9d forms of 17 kD requires specific cellular proteases. In contrast, IL-1Ra evolved with a signal peptide and is readily transported out of the cells and termed secreted IL-1Ra (sIL-1Ra).
IL-1 Receptor and Ligands.
The receptors and ligands of the IL-1 pathway have been well defined (for review, see Dinarello, C. A., FASEB J. 8:1314-1325 (1994); Sims, J. E. et al., Interleukin-1 signal transduction: Advances in Cell and Molecular Biology of Membranes and Organelles, Vol.3, JAI Press, Inc., Greenwich, Conn. (1994), pp. 197-222). Three ligands, IL-1xcex1, IL-1xcex2, and IL-1 receptor antagonist (IL-1Ra) bind three forms of IL-1 receptor, an 80-kDa type I IL-1 receptor (IL-IR1) (Sims, J. E. et al., Science 241:585-589 (1988)), a 68-kDa type II IL-1 receptor (IL-1RII) (McMahan, C. J. et al., EMBO J. 10:2821-2832 (1991)), and a soluble form of the type II IL-1R (sIL-1RII) (Colotta, F. et al., Science 261:472-475 (1993)).
The interactions between the IL-1 ligands and receptors play an essential role in the stimulation and regulation of the IL-1 -mediated host response to injury and infection. Cells expressing IL-1RI and treated with IL-1xcex1 or IL-1xcex2 respond in several specific ways, including stimulating nuclear localization of the rel-related transcription factor, NF-xcexaxcex2 (for review, see Thanos, D. and Maniatis, T., Cell 80:529-532 (1996)), activation of protein kinases of the mitogen-activated protein kinase superfamily that phosphorylate residue threonine 669 (Thr-669) of the epidermal growth factor receptor (EGFR) (Guy, G. R. et al., J. Biol. Chem. 267:1846-1852(1992); Bird, T. A. et al., J. Biol. Chem. 268:22861-22870(1991); Bird, T. A. et al., J. Biol. chem. 269:31836-31844 (1994)), and stimulation of transcription of the IL-8 gene (Mukaida, N. et al., J. Biol. chem. 265:21128-21133 (1990)).
IL-1RI-like Family.
Many proteins from diverse systems show homology to the cytoplasmic domain of the IL-1RI. This expanding IL-1RI-like family includes mammalian proteins, Drosophila proteins, and a plant (tobacco) protein. (Gay, N. J. and Keith, F. J., Nature 351:355-356 (1991); Hashimoto, C. et al., Cell 52:269-279 (1988); Schneider, D. S. et al., Genes and Dev. 5:797-807 (1991); Edon, E. et al., Development 120:885-899 (1994); Mitchan, J. L. et al., J. Biol. Chem 271:5777-5782 (Mar. 8, 1996)).
The mammalian IL-1RI-like receptor family members include a murine protein MyD88 (Lord, K. A. et al., Oncogene 5:1095-1097 (1990)) and a human gene, rsc786 (Nomura, N. et al., DNA Res. 1:27-35 (1994)). Another murine receptor member, T1/ST2, was previously characterized as a novel primary response gene expressed in BALB/c-3T3 cells (Klemenz, R. et al., Proc. Natl. Acad. Sci. USA 86:5708-5712 (1989); Tominaga, S., FEBS Lett. 258:301-304 (1989); Tominga, S. et al., FEBS Lett. 318:83-87 (1993)). The transmembrane protein mulL-1R AcP (Greenfeder, S. A. et al., J. Biol. Chem. 270:13757-13765 (1995)) has homology to both the type I and type II IL-1R. IL-1R AcP has recently been shown to increase the affinity of IL-1RI for IL-1xcex2 and may be involved in mediating the IL-1 response.
T1 Receptors.
T1/ST2 receptors (hereinafter, xe2x80x9cT1 receptorsxe2x80x9d), as a member of the IL-1 receptor family (Bergers, G., et al., EMBO J. 13:1176 (1994)), have various homologs in different species. In the rat, it is called Fit-1, an estrogen-inducible, c-fos-dependent transmembrane protein that shares 26% to 29% amino acid homology to the mouse IL-1RI and II, respectively. In the mouse, the Fit-1 protein is called ST2 and in the human it is called T1. The organization of the two IL-1 receptors and the Fit-1/ST2/T1 genes indicates they are derived from a common ancestor (Sims, J. E., et al., Cytokine 7:483 (1995)). Fit-1 exists in two forms: a membrane form (Fit-1M) with a cytosolic domain similarly to that of the IL-1RI and Fit-1S, which is secreted and composed of the extracellular domain of Fit-M.
In many ways, these two forms of the Fit-1 protein are similar to those of the membrane-bound and soluble IL-1RI. It has been shown that the IL-1sRI is derived from proteolytic cleavage of the cell-bound form (Sims, J. E., et al., Cytokine 7:483 (1995)). On the other hand, the Fit-1 gene is under the control of two promoters, which results in two isoforms coding for either the membrane or soluble form of the receptor. Two RNA transcripts result from alternative RNA splicing of the 3xe2x80x2 end of the gene. Although IL-1xcex2 binds weakly to Fit-1 and does not transduce a signal (Reikerstorger, A., et al., J. Biol. Chem. 270:17645 (1995)), a chimeric receptor consisting of the extracellular murine IL-1RI fused to the cytosolic Fit-1 transduces an IL-1 signal (Reikerstorger, A., et al., J. Biol. Chem. 270:17645 (1995)). The cytosolic portion of Fit-1 align with GTPase-like sequences of IL-1RI (Hopp, T. P., Protein Sci. 4:1851 (1995)) (see below).
IL-1 Production in Various Disease States.
Increased IL-1 production has been reported in patients with various viral, bacterial, fungal, and parasitic infections; intravascular coagulation; high-dose IL-2 therapy; solid tumors; leukemias; Alzheimer""s disease; HIV-1 infection; autoimmune disorders; trauma (surgery); hemodialysis; ischemic diseases (myocardial infarction); noninfectious hepatitis; asthma; UV radiation; closed head injury; pancreatitis; periodontitis; graft-versus-host disease; transplant rejection; and in healthy subjects after strenuous exercise. There is an association of increased IL-1xcex2 production in patients with Alzheimer""s disease and a possible role for IL-1 in the release of the amyloid precursor protein (Vasilakos, J. P., et al., FEBS Lett. 354:289 (1994)). However, in most conditions, IL-1 is not the only cytokine exhibiting increased production and hence the specificity of the IL-1 findings as related to the pathogenesis of any particular disease is lacking. In various disease states, IL-1, but not IL-1xcex1, is detected in the circulation.
IL-1 in Therapy.
Although IL-1 has been found to exhibit many important biological activities, it is also found to be toxic at doses that are close to therapeutic dosages (Dinarello, C. A., Blood 87:2095-2147 (Mar. 15,1996)). In general, the acute toxicities of either isoform of IL-1 were greater after intravenous compared with subcutaneous injection. Subcutaneous injection was associated with significant local pain, erythema, and swelling (Kitamura, T., and Takaku, F., Exp. Med. 7:170 (1989); Laughlin, M. J., Ann. Hematol. 67:267 (1993)). Patients receiving intravenous IL-1 at doses of 100 ng/kg or greater experienced significant hypotension. In patients receiving IL-1xcex2 from 4 to 32 ng/kg subcutaneously, there was only one episode of hypotension at the highest dose level (Laughlin, M. J., Ann. Hematol. 67:267 (1993)).
Contrary to IL-1-associated myelostimulation in patients with normal marrow reserves, patients with a plastic anemia treated with 5 daily doses of IL-1xcex1 (30 to 100 ng/kg) had no increases in peripheral blood counts or bone marrow cellularity (Walsh, C. E., et al., Br. J. Haematol 80:106 (1992)). IL-1 has been administered to patients undergoing various regiments of chemotherapy to reduce the nadir of neutropenia and thrombocytopenia.
Daily treatment with 40 ng/kg IL-1xcex1 from day 0 to day 13 of autologous bone marrow or stem cells resulted in an earlier recovery of neutropenia (median, 12 days; P less than 0.001) (Weisdorf, D., et al., Blood 84:2044 (1994)). After 14 days of treatment, the bone marrow was significantly enriched with committed myeloid progenitor cells. Similar results were reported in patients with AML receiving 50 ng/kg/d of IL-1xcex2 for 5 days starting at the time of transplantation with purged or nonpurged bone marrow (Nemunaitis, J., et al., Blood 83:3473 (1994)). Injecting humans with low doses of either IL-1xcex1 or IL-1xcex2 confirms the impressive pyrogenic and hypotension-inducing properties of the molecules.
Amelioration of Disease using Soluble IL-1Receptors.
Administration of murine IL-1sRI to mice has increased the survival of heterotopic heart allografts and reduced the hyperplastic lymph node response to allogeneic cells (Fanslow, W. C., et al., Science 248:739 (1990)). In a rat model of antigen-induced arthritis, local instillation of the murine IL-1sRI reduced joint swelling and tissue destruction (Dower, S. K., et al., Therapeutic Immunol. 1:113 (1994)). These data suggest that the amount of IL-1sRI administered in the normal, contralateral joint was acting systemically. In a model of experimental autoimmune encephalitits, the IL-1sRI reduced the severity of this disease (Jacobs, C. A., et al., J. Immunol. 146:2983 (1991)).
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a human T1 receptor-(T1R-)like ligand II polypeptide having the amino acid sequence in FIGS. 1A-1B (SEQ ID NO:2). The T1R-like ligand II contains an open reading frame encoding a polypeptide of about 229 amino acid residues including an N-terminal methionine, a leader sequence of about 26 amino acid residues, an extracellular mature domain of about 168 residues, a transmembrane domain of about 23 residues and an intracellular domain of about 12 amino acid residues, and a deduced molecular weight of about 26 kDa. The 203 amino acid sequence of the expected mature T1R-like ligand II protein is shown in SEQ ID NO:2 (amino acid residues 1-203).
The invention also provides isolated nucleic acid molecules encoding an T1R-like ligand II having an amino acid sequence encoded by the cDNA of the clone deposited as ATCC Deposit No. 97655 on Jul. 12, 1996. Preferably, the nucleic acid molecule will encode the mature polypeptide encoded by the above-described deposited cDNA.
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the T1R-like ligand II polypeptide having the complete amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequence encoding the T1R-like ligand II polypeptide having the complete amino acid sequence in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) a nucleotide sequence encoding the mature T1R-like ligand II polypeptide having the amino acid sequence at positions from about I to about 203 in SEQ ID NO:2; (d) a nucleotide sequence encoding the T1R-like ligand II polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97655; (e) a nucleotide sequence encoding the mature T1R-like ligand II polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97655; and (f) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), or (e) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d), (e), or (f), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), or (f), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a T1R-like ligand II polypeptide having an amino acid sequence in (a), (b), (c), (d), or (e), above.
The present invention also relates to recombinant vectors which include the isolated nucleic acid molecules of the present invention, host cells containing the recombinant vectors, and the production of T1R-like ligand II polypeptides or fragments thereof by recombinant techniques.
The invention further provides an isolated T1R-like ligand II polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the T1R-like ligand II polypeptide having the complete 229 amino acid sequence, including the leader sequence shown in SEQ ID NO:2; (b) the amino acid sequence of the T1R-like ligand II polypeptide having the complete 229 amino acid sequence, including the leader sequence shown in SEQ ID NO:2 but minus the N-terminal methionine residue; (c) the amino acid sequence of the mature T1R-like ligand II polypeptide (without the leader) having the amino acid sequence at positions 1 to 203 in SEQ ID NO:2; (d) the amino acid sequence of the T1R-like ligand II polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC Deposit No. 97655; and (e) the amino acid sequence of the mature T1R-like ligand II polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97655. The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 90% identical, and more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a T1R-like ligand II polypeptide having an amino acid sequence described in (a), (b), (c), (d), or (e), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a T1R-like ligand II polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a T1R-like ligand II polypeptide having an amino acid sequence described in (a), (b), (c), (d), or (e) above.
The invention also relates to fragments of the above-described polypeptides. Preferred polypeptide fragments according to the present invention include a polypeptide comprising: the mature polypeptide (amino acid residues from about 1 to about 203 in SEQ ID NO:2), the extracellular domain (amino acid residues from about 1 to about 168 in SEQ ID NO:2), the transmembrane domain (amino acid residues from about 169 to about 191 in SEQ ID NO:2), the intracellular domain (amino acid residues from about 192 to about 203 in SEQ ID NO:2), or the extracellular and intracellular domain with all or part of the transmembrane domain deleted.
In addition, the invention provides for fusion polypeptides of T1R-like ligand II which may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling and/or codon-shuffling.
The invention further provides for proteins containing polypeptide sequences encoded by the polynucleotides of the invention. The proteins may be in the form of monomers or multimers. The preparation of these proteins and compositions (preferably pharmaceutical compositions) containing these proteins are also provided.
In another embodiment, the invention provides transgenic animals which express the polypeptides and proteins of the invention.
In yet another embodiment, chromosome assays are provided which allow for chromosome identification. Nucleic acids of the invention can be used to specifically target and hybridize to a particular location on an individual human chromosome. Once a sequence has been mapped to a precise chromosome location, the physical position of the sequence on the chromosome can be correlated with genetic map data.
In another embodiment, the invention provides for antisense and ribozyme antagonists of T1R-like ligand II.
It is believed that biological activities of the T1R-like ligand II of the present invention may be similar to the biological activities of the T1R ligand and IL-1. Significantly, higher or lower levels of T1R-like ligand II may be detected in tissues or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having a T1R ligand- or IL-1-related disorder, relative to a xe2x80x9cnormalxe2x80x9d T1R-like ligand II gene expression level, i.e., the expression level in tissue or bodily fluids from an individual not having the T1R ligand- or IL-1-related disorder. Thus, detecting expression of T1R-like ligand II gene expression according to the present invention is a diagnostic marker. Accordingly, the invention provides for diagnostic kits used to detect levels of T1R-like ligand II expression.
The invention also provides methods for producing and isolating antibodies that bind specifically to an T1R-like ligand II polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described herein.
The invention is further related to a method for treating an individual in need of an increased or decreased level of T1R-like ligand II activity in the body, comprising administering to such an individual a composition comprising a T1R-like ligand II polypeptide or an inhibitor thereof.
As such, pharmaceutical compositions of T1R-like ligand II are provided. Formulations of T1R-like ligand II are also provided as are methods for administering therapeutic doses of T1R-like ligand II polynucleotides, polypeptides, antibodies, agonists, antagonists and/or fragments and variants thereof.
Finally, the invention provides for methods of using the polynucleotides encoding T1R-like ligand II polypeptides, antibodies, agonists, antagonists, and/or fragments and variants thereof, in gene therapy.